The phytohormone cytokinin (CK) positively regulates the activity and function of the shoot apical meristem (SAM), which is a major parameter determining seed production. The rice (Oryza sativa L.) Gn1a/OsCKX2 (Grain number 1a/Cytokinin oxidase 2) gene, which encodes a cytokinin oxidase, has been identified as a major quantitative trait locus contributing to grain number improvement in rice breeding practice. However, the molecular mechanism of how the expression of OsCKX2 is regulated in planta remains elusive. Here, we report that the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST) directly regulates OsCKX2 expression in the reproductive meristem. DST-directed expression of OsCKX2 regulates CK accumulation in the SAM and, therefore, controls the number of the reproductive organs. We identify that DST(reg1), a semidominant allele of the DST gene, perturbs DST-directed regulation of OsCKX2 expression and elevates CK levels in the reproductive SAM, leading to increased meristem activity, enhanced panicle branching, and a consequent increase of grain number. Importantly, the DST(reg1) allele provides an approach to pyramid the Gn1a-dependent and Gn1a-independent effects on grain production. Our study reveals that, as a unique regulator of reproductive meristem activity, DST may be explored to facilitate the genetic enhancement of grain production in rice and other small grain cereals.

SUMMARYAbscisic acid (ABA) and cytokinin are key hormones controlling plant development. How ABA and cytokinin interplay to control the transition from a dry seed into a young seedling remains elusive. Here we undertook a gain-of-function genetic screen to identify ABA-insensitive mutants during seed germination in Arabidopsis using an estradiol-inducible approach. In the presence of estradiol, one of these mutants gim1 (germination insensitive to ABA mutant 1) exhibited an elevated level of cytokinin that was attributed to the estradiolinduced expression of AtIPT8 that encodes an isopentenyltransferase for the biosynthesis of cytokinins. Our data on OE-2 and Com-1 transgenic plants carrying the ectopically expressing AtIPT8 gene indicated that the elevation of cytokinin level was responsible for the ABA-insensitivity of gim1 seed germination. Further analyses on alterations of gene transcriptomes in the gim1 mutant demonstrated that the expression of some ABA-inducible genes, including ABI5, was reduced, and could not be restored by exogenous ABA treatment. Moreover, we also failed to observe the ABA-mediated repression of a family of cytokinin signal transducers and transcription repressors called type-A ARR4, ARR5 and ARR6 in the gim1 seedlings. Further analysis demonstrated that type-A ARR4, ARR5 and ARR6 could negatively regulate ABI5 expression, and the physical interaction of ABI5 and type-A ARR4, ARR5 and ARR6 proteins was detected. In summary, our study suggests that the interaction of ABA and cytokinin during seed germination and seedling growth can be mediated by the interplay of transcriptional regulators in Arabidopsis.

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In this study, a sensitive assay of cytokinins was developed using polymer monolith microextraction/ hydrophilic interaction chromatography/electrospray ionization-tandem mass spectrometry (PMME/ HILIC/ESI-MS/MS). The extraction was realized on a poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-ethylene dimethacrylate) (poly(AMPS-co-EDMA)) capillary monolith and the subsequent separation was carried out on a Luna silica column. Several parameters of PMME and HILIC were optimized to obtain the optimum results. After optimizing the extraction conditions, 10 mM ammonium formate of pH 2.5 was chosen as the matrix solution to obtain the highest extraction efficiency. The MS sensitivity for cytokinins investigated could be enhanced 3-fold by the use of hydrophilic interaction chromatography with the mobile phase of 85% acetonitrile with 0.01% (v/v) formic acid and 15% water with 0.01% (v/v) formic acid, when compared to the use of conventional reversed phase liquid chromatography (RPLC). Good linearities were obtained for five cytokinins with correlation coefficients (R 2 ) > 0.9962. The LODs (S/N ¼ 3) for the targets were found to be 0.0028-0.068 ng mL À1 . Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations (RSDs) less than 12.7% and the recoveries in plant samples ranged from 70.3% to 113.3%. The method was applied to the determination of cytokinins in Oryza sativa, Arabidopsis thaliana and oil seed rape tissues.

Electrochemical synthesis has been rapidly developed over the past few years, while a vast majority of the reactions proceed through a radical pathway. Understanding the properties of radical intermediates is crucial in the mechanistic study of electrochemical transformations and will be beneficial for developing new reactions. Nevertheless, it is rather difficult to determine the “live” radical intermediates due to their high reactivity. In this work, the formation and structure of sulfonamide N-centered radicals have been researched directly by using the time-resolved electron paramagnetic resonance (EPR) technique under electrochemical conditions. Supported by the EPR results, the reactivity of N-centered radicals as a mediator in the hydrogen atom transfer (HAT) approach has been discussed. Subsequently, these mechanistic study results have been successfully utilized in the discovery of an unactivated C­(sp3)–H arylation reaction. The kinetic experiments have revealed the rate-determined step is the anodic oxidation of sulfonamides.